The following includes information that may be useful in understanding the present inventions. It is not an admission that any of the information provided herein is prior art, or relevant, to the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced is prior art.
Coronary heart disease is the leading cause of death in most western countries. Mortality rates for cardiovascular disease have been reported to vary from 29 (in males, 24 for females) per 100,000 in Canada up to 213 (males, females 154) in the Russian Federation. For other Western countries the rate is 31-55 (43-26). Approximately half the deaths attributable to stroke are the result of medical complications, such as pneumonia and sepsis, and half are attributable to neurological complications, such as new cerebral infarction and cerebral edema. Brott, T. and Bogousslaysky, J., New Engl. J. Med. 343: 710-722 (2000).
Strokes are the third leading cause of death in developed countries and have a devastating impact on public health. About 700,000 new stroke cases occur in America every year (American Stroke Association). About 25% of stroke sufferers die as a result of the stroke or its resulting complications. Additionally, almost 50% of stroke victims have moderate to severe health impairments and long-term disabilities. Although the incidence of ischemic stroke has declined over the past 20 years, the mean age of the population has risen, resulting in a continual increase in the absolute number of strokes. Recent projections indicate that by the year 2050, more than 1 million strokes will occur each year in the United States. Strokes are generally the result of several underlying conditions that decrease the flow of blood to the brain and cause disability or death. Approximately 85 percent of strokes are ischemic in nature (blood clot or blockage of a blood vessel). Foulkes M R, et al., Stroke; 19:547-54 (1988). An ischemic stroke can be caused by a blood clot that forms inside the artery of the brain (thrombotic stroke), or by a clot that forms somewhere else in the body and travels to the brain (embolic stroke), with thrombotic strokes representing about 52% of all ischemic strokes. Thrombotic strokes are generally a result of atherosclerosis (where blood vessels become clogged with a buildup of fatty deposits, calcium, and blood clotting factors such as fibrinogen and cholesterol).
Inflammation is a multifactorial process, and is manifest in many diseases, disorders, and conditions which have enormous cumulative health consequences. Inflammatory diseases, including rheumatoid arthritis, lupus, psoriasis, multiple sclerosis and asthma remain a major cause of mortality and morbidity worldwide. Autoimmune diseases are also associated with inflammation and on the rise, reportedly affecting more than 50 million people in the U.S. In many autoimmune diseases, cell, tissue, joint and organ damage results from the uncontrolled activation of a immense array of inflammatory pathways. Rheumatoid arthritis (RA) is one such chronic inflammatory disease characterized by inflammation of the joints, leading to swelling, pain, and loss of function. RA affects at least an estimated 2.5 million people in the United States, and is caused by a combination of events including an initial infection or injury, an abnormal immune response, and genetic factors. Any one of at least 80 different autoimmune diseases can result when the immune system becomes unregulated and attacks healthy tissue.
Connexins, also known as gap junction proteins, are four-pass transmembrane proteins with cytoplasmic C and N termini. Six connexins combine together to form a hemichannel called a “connexon.”
Gap junctions are structures that provide direct cell-to-cell communication. The gap junction is composed of two connecting connexons, one contributed by each of the abutting cells that upon docking form a functional gap junction.
As they are being translated by ribosomes, connexins are inserted into the membrane of the endoplasmic reticulum. Bennett M V, Zukin R S. Electrical coupling and neuronal synchronization in the Mammalian brain. Neuron. 2004 Feb. 19; 41(4):495-511. There they gather to form hemichannels (connexons), which are carried to the cell membrane in vesicles and diffuse through the membrane until they meet a hemichannel from the other cell, with which they can dock to form a channel. Id. Molecules on a connexin allow it to “recognize” the other connexins in their hemichannel and those of the other cell's hemichannel, and cause correct alignment and formation of the channel. Kandel E R, Schwartz J H, Jessell T M. Principles of Neural Science, 4th ed., pp. 178-180. McGraw-Hill, New York (2000).
Connexin proteins have a common transmembrane topology, with four alpha-helical transmembrane domains, two extracellular loops, a cytoplasmic loop, and cytoplasmic N- and C-terminal domains. The sequences are most conserved in the transmembrane and extracellular domains, yet many of the key functional differences between connexins are determined by amino-acid differences in these largely conserved domains. Each extracellular loop contains three cysteines with invariant spacing (save one isoform) that are required for channel function. The junctional channel is composed of two end-to-end hemichannels, each of which is a hexamer of connexin subunits. In junctional channels, the cysteines in the extracellular loops form intra-monomer disulfide bonds between the two loops, not intermonomer or inter-hemichannel bonds. The end-to-end homophilic binding between hemichannels is via non-covalent interactions. Mutagenesis studies suggest that the docking region contains beta structures, and may resemble to some degree the beta-barrel structure of porin channels. The two hemichannels that compose a junctional channel are rotationally staggered by approximately 30 degrees relative to each other so that the alpha-helices of each connexin monomer are axially aligned with the alpha-helices of two adjacent monomers in the apposed hemichannel.
Each connexon or hemichannel in the membrane should, under normal conditions, remain closed until it docks with a connexon of a neighboring cell. However, the inventors believe that when a cell expressing a hemichannel is subjected to a stress (e.g. physiological, mechanical, etc.) hemichannels can open even when they are not docked. The inhibition of extracellular hemichannel communication includes the inhibition of the flow of small molecules through an open hemichannel to and from an extracellular or periplamic space. While not intending to be bound by or limited to any mechanism, modes of action include blocking (partial or complete) of the hemichannel, triggering internalization of the connexon which is then removed from the membrane, inducing a conformational change in the connexin proteins to bring about closure of the connexon, and masking or binding to sites involved in triggering channel opening (such calcium binding sites).
Antisense (AS) nucleotides to connexins and uses thereof have been described. See WO00/44409 to Becker et al., filed Jan. 27, 2000, “Formulations Comprising Antisense Nucleotides to Connexins.”